Phosphoprotein molecular profiling is an important component of the emerging field of individualized cancer therapy. Molecularly targeted anti-cancer therapeutics often involves the mitigation or modulation of defective kinase signaling networks. Elucidation of deranged signaling networks within tumors offers tremendous promise as a means to individualize molecular targeted cancer therapy and to identify entirely new targets for therapeutic intervention. Evidence from molecular signature/transcript studies using gene microarrays (nucleic acid analysis) suggests that each patient's tumor may have a unique genetic portrait. Nevertheless, while gene microarrays can provide important information about somatic genetic taxonomy, they are unable to provide an effective recapitulation of the drug targets themselves, which are embodied in the post-translational and fluctuating signaling molecular network events that occur at the proteomic level. The phosphorylation, or activation state, of kinase-driven signal networks contains important information concerning both disease pathogenesis and the ongoing state of kinase-associated therapeutic targets. It is for this reason that modulation of ongoing cellular kinase activity represents one of the most rapidly growing arenas in new drug discovery. Identification of specific phosphoprotein signaling aberrations can be used, e.g., for the development of targeted therapies for patients with lung, breast, colon, or other cancer. Profiling the tumor phosphoproteome using human tumor biopsy specimens is an important component of the perceived upcoming revolution of individualized cancer therapy.
While proteomic molecular profiling offers tremendous promise to change the practice of oncology, the fidelity of the data obtained from a diagnostic assay applied to tissue must be monitored and ensured; otherwise, a clinical decision may be based on incorrect molecular data. To date, clinical preservation practices routinely rely on protocols that are decades old, such as formalin fixation, and are designed to preserve specimens for histologic examination. Tissue is generally procured for pathologic examination in three main settings: a) surgery in a hospital-based operating room, b) biopsy conducted in an outpatient clinic, and c) image-directed needle biopsies or needle aspirates conducted in a radiologic suite. Currently, tissue is generally snap-frozen in order to perform proteomic studies. In the real world of a busy clinical setting, it may be impossible to immediately preserve procured tissue in liquid nitrogen. Moreover, the time delay from patient excision to pathologic examination and molecular analysis is often not recorded and may vary from 30 minutes to many hours depending on the time of day, the length of the procedure, and the number of concurrent cases.
FIG. 1 depicts the two categories of variable time periods that define the stability intervals for tissue procurement (e.g. from human tissue). Time point A is defined as the moment that tissue is excised from the patient and becomes available ex vivo for analysis and processing. The post excision delay time, or EDT, is the time from time point A to the time that the specimen is placed in a stabilized state, e.g., immersed in fixative or snap-frozen in liquid nitrogen, herein called time point B. Given the complexity of patient-care settings, during the EDT the tissue may reside at room temperature in the operating room or on the pathologist's cutting board, or it may be refrigerated in a specimen container. The second variable time period is the processing delay time, or PDT. At the beginning of this interval the tissue is immersed in a preservative composition or stored in a freezer. At the end of this interval, time point C, the tissue is subject to processing for molecular analysis. In addition to the uncertainty about the length of these two time intervals, a host of known and unknown variables can influence the stability of tissue molecules during these time periods. These include 1) temperature fluctuations prior to fixation or freezing, 2) preservative chemistry and rate of tissue penetration, 3) size of the tissue specimen, 4) extent of handling, cutting, and crushing of the tissue, 5) fixation and staining prior to microdissection, 6) tissue hydration and dehydration, and 7) the introduction of phosphatases or proteinases from the environment at any time.
There is a need for methods to collect and preserve (fix and stabilize) proteins, including post-translationally modified proteins, such as phosphoproteins (e.g. within about four hours of corporal extraction), and for methods to monitor the status (e.g. phosphorylation state) of proteins during the EDT and PDT periods.